Activation of the mapk signal cascade components phosphorylation involved in the formation of the G0-positive tumor phenotype

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Abstract

Introduction. Among the heterogeneous population of tumor cells, there are so-called dormant and senescent cells located in the G0 phase of the cell cycle. The transition to the G0 phase is a stress response mediated, for example, by treatment with chemotherapeutic drugs. The functioning of such cells is associated with the development of non-response.

The aim of the study. G0-positive skin melanoma cells modulation with subsequent assessment of the MARK signal cascade molecules, including the main tumor suppressor p53.

Material and methods. Skin melanoma cells were incubated with the cytostatic drug dacarbazine to induce the level of G0-positive cells. Total RNA extracted from cells was used for transcriptome analysis, after which the level of phosphorylation of MARK key molecules was evaluated. By immunocytochemistry (ICC) and real-time PCR (PCR-RT) the activity of tumor suppressor p53 was analyzed.

Results. As a result of the G0-positive cells level modulation, the MARK signal cascade is among the signaling pathways with the largest number of genes with altered expression. Significantly increased the number of phosphorylated proteins JNK, p70S6K, MEK, RSK1 and RSK2, as well as protein p53, capable of forming a senescent phenotype of tumor cells.

Conclusion. When the level of G0-positive skin melanoma cells is modulated by the cytostatic drug dacarbazine, phosphorylation of the MARK signaling cascade components involved in the formation of the G0-positive tumor phenotype increases.

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About the authors

Aleksandra R. Esimbekova

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Author for correspondence.
Email: aleksandra.esimbekova.96@mail.ru
ORCID iD: 0000-0001-6363-5941

PhD student of the Department of Pathophysiology

Russian Federation, P. Zeleznyak street, 1, Krasnoyarsk, 660022

Ekaterina Z. Lapkina

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: e.z.lapkina@mail.ru
ORCID iD: 0000-0002-7226-9565

Associate Professor of the Department of Pharmacy, Candidate of biological sciences

Russian Federation, P. Zeleznyak street, 1, Krasnoyarsk, 660022

Tatiana G. Ruksha

Professor V.F. Voino-Yasenetsky Krasnoyarsk State Medical University

Email: tatyana_ruksha@mail.ru
ORCID iD: 0000-0001-8142-4283

Head of the Department of Pathophysiology, Doctor of medical sciences, Professor

Russian Federation, P. Zeleznyak street, 1, Krasnoyarsk, 660022

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Supplementary files

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2. Fig. 1. Results of microarray assay of SK-MEL-2 melanoma cells. Heatmap showing differentially altered MAPK pathway transcripts in following dacarbazine treatment

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3. Fig. 2. The cytostatic drug dacarbazine causes activation of MAPK/ERK kinase phosphorylation. The graph shows the normalized average pixel density of protein levels in lysates obtained from SK-MEL-2 cells using an antibody membrane containing 17 duplicated antibodies against various MARK signaling molecules, 4 positive and 3 negative controls. Signals were analyzed using Image Lab (Bio Rad) software. Values from duplicate points were averaged and plotted on a graph

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4. Fig. 3. Tumor suppressor p53 activation in SK-MEL-2 skin melanoma cells after induction of the G0-positive cells level following dacarbazine treatment; a – tp53 expression levels according to the real-time PCR; b – p53 protein levels according to the immunocytochemistry with antibodies p53 and DAPI

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